1. Field of the Invention
The invention relates to cloning vectors useful for the expression of T-cell variable domains, to bacterial cells transformed by the vectors and to methods of producing T-cell variable domains in a prokaryotic host cell, either as single domains or as single chain heterodimers.
2. Description of Related Art
The production of single or heterodimeric T-cell receptor variable domains is of interest for purposes of studying T-cell receptor interaction with antigens and possibly developing approaches to therapies for autoimmune diseases and cancer. An important goal of molecular biology is a detailed understanding at the molecular level of the binding of T-cell receptors to cognate peptide-major histocompatibility complexes. This will be a step in the development, for example, of immunotherapy for T-cell mediated autoimmune disease. Despite this interest and the potential applications arising from the study of T-cell receptor domains, no methods are available for the production of only single T-cell receptor domains, nor has expression and secretion in prokaryotic hosts been successful.
The majority of T cells recognize antigenic peptides bound to class I or II proteins of the major histocompatibility complex (MHC) and are thus xe2x80x9cMHC restrictedxe2x80x9d. The recognition of peptide-MHC complexes is mediated by surface-bound T-cell receptors (TCRs). These receptors are comprised of various heterodimeric polypeptides, the majority of which are xcex1 and xcex2 polypeptides. A minor population (1-10%) of mature T-cells bear T-cell receptors (TCRs) comprising xcex4xcex3 heterodimers (Borst et al., 1987; Brenner et al., 1986).
Several composite dimeric species incorporating the xcex1 and xcex2 polypeptides have been produced in various systems. TCR xcex1xcex2 heterodimers have been expressed as phosphatidyl-inositol linked polypeptides (Lin et al., 1990) or TCR-immunoglobulin chimeras (Gregoire et al., 1991) in mammalian transfectomas. The production of Vxcex1Cxcexa homodimers (Mariuzza and Winter, 1989) and Vxcex2-Cxcex2 monomers (Gascoigne, 1990) in mammalian cells has also been described. The expression and secretion of immunoglobulin VH domains (Ward et al., 1989), Fv fragments (Skerra and Pluckthum, 1988; Ward et al., 1989) and Fab fragments (Better et al., 1988) has been reported. Molecular modeling analyses indicate that there are structural similarities between immunoglobulin Fab fragments and the extracellular domains of TCRs (Novotny et al., 1986; Chothia et al., 1988). Several expression systems for the production of recombinant TCRs in mammalian cell transfectomas have been documented but successful expression and secretion of these proteins in a prokaryotic host has not been reported.
Despite apparent expression of a single chain anti-fluorescein TCR in E. coli (Novotny et al., 1991), the product could not be isolated from the periplasm even though the leader sequence had been cleaved from the N-terminus of the recombinant protein. The single chain TCR was relatively insoluble, requiring the use of genetic manipulation to replace five of the xe2x80x9cexposedxe2x80x9d hydrophobic residues with relatively hydrophilic residues.
No methods are presently available for the production of single or heterodimeric T-cell receptor variable domains as secreted proteins. If available, such species would have potential use in the induction of antibodies as protective vaccines, for the therapy of autoimmune disease, and antibodies for targeting idiotypes (T-cell) or T-cell leukemias. Additionally, secretion of T-cell receptor domains from bacterial cell hosts should provide a convenient, economically attractive and rapid route for production of recombinant T-cell receptors.
Advantages of the production of the TCR variable domains in E. coli compared with expression of phosphatidyl-inositol linked heterodimers and TCR-immunoglobulin chimeras in mammalian cells are the following: (1) E. coli (and other prokaryotic hosts) grow much faster; thus, results of genetic manipulation of the fragments can be analyzed more quickly, (2) use of E. coli is much cheaper than mammalian hosts, (3) production of only the TCR variable domains in mammalian hosts has not been reported. For raising anti-idiotypic antibodies (which recognize variable domains only), this is particularly significant.
TCR fragments have been produced in mammalian cells but they are relatively large. Smaller size TCR segments may allow more rapid structural resolution using such techniques as NMR and X-ray crystallography. Since the variable domains are the regions which interact with peptide-MHC complexes, these regions of TCRs are of considerable interest. Additionally, the use of variable domains alone in immunization should result in the production of anti-variable domain antibodies. Such antibodies are expected to be particularly desirable for use in therapy and diagnosis since they block the interaction of the TCR with antigen and, due to the variable nature of the Vxcex1/Vxcex2 domains or other domains such as Vxcex3 and Vxcex4, are specific for subsets of T-cells. Large TCR fragments, such as those that can be expressed from mammalian cells, result in production of antibodies not only against the variable domains, but also against the TCR constant domains, (if present in the construct) and/or the immunoglobulin domains (if present in the construct). There would therefore be distinct advantages in having smaller variable domain TCR fragments available, particularly for immunization since any immune response generated is likely to be directed to particular regions of interest, i.e., the V domains.
The present invention seeks to address one or more of the foregoing problems associated with expression and secretion of T-cell receptor variable domains in a prokaryotic host cell. Recombinant Vxcex1, Vxcex2 and single chain Vxcex1Vxcex2 heterodimers have been produced in gram-negative hosts transformed with vectors containing DNA encoding one or more T-cell receptor variable domains. The T-cell receptor domains are efficiently secreted in E. coli or S. marcescens. Only the TCR proteins expressed in E. coli have been characterized by CD. These products contain a high proportion of xcex2-sheet structure indicative of a native structure. Murine T-cell Vxcex1 and Vxcex2 domains have been expressed and isolated in yields up to milligram quantities per liter of bacterial culture. Single T-cell variable domains (Vxcex1 and Vxcex2) and single chain (sc) Vxcex1Vxcex2 heterodimers have been produced employing the disclosed vectors.
The recombinant plasmids or expression vectors of the invention are particularly adapted for expression of T-cell receptor domains in transformed prokaryotic host cells. The recombinant plasmids comprise a DNA segment coding for one or more T-cell receptor variable domains. Any of a number of variable domains may be included but preferred domains are the Vxcex1 and Vxcex2. Murine T-cell receptor domain Vxcex1Vxcex2 heterodimer, derived from the 1934.4 hybridoma (Wraith et al., 1989) is particularly preferred. Segments of the Vxcex1 or Vxcex2 domains as well as other variable domains such as Vxcex3Vxcex4, constant domains, Cxcex1, Cxcex21, Cxcex22, Cxcex4, Cxcex3 or immunoglobulin CH1, CH2 and CH3 domains, etc. may also be employed. It is also contemplated that variations of T-cell receptor variable domains also fall within the scope of the invention. Such variations may arise from mutations such as point mutations and other alterations affecting one or more amino acids or the addition of amino acids at the N or C termini. While the invention has been illustrated with murine T-cell receptors, similar strategies are applicable to the receptor domains from other species, including rat, man and other mammals.
Other DNA segments may also be included linked to the variable domains described, for example, one or more recombinant T-cell receptor variable domains of one or more specificities linked to TCR constant domains, immunoglobulin constant domains, or bacteriophage coat protein genes. Once expressed, any of the products herein could be radiolabelled or fluorescently labeled, or attached to solid supports, including sepharose or magnetic beads or synthetic bilayers such as liposomes. The products could also be linked to carrier proteins such as bovine serum albumin. The TCR V domains, or V domains linked to other proteins (such as constant domains), could also be linked synthetically to co-receptors such as the extracellular domains of CD4 or CD8. This could increase the avidity of the interaction of the TCR fragment with cognate peptide MHC complexes.
Cloning vectors are included in one aspect of the present invention. The vectors include a leader sequence, preferably pelB (Better et al., 1988), although other leader sequences may be used, for example, alkaline phosphatase (phoA) or ompA. In a preferred embodiment, the pelB leader segment is modified with a unique restriction site, such as NcoI, allowing insertion of TCR variable domain genes. Introduction of such restriction sites is a convenient means of cloning in a DNA segment in the same reading frame as the leader sequence.
Modification of the leader sequence DNA may be achieved by altering one or more nucleotides employing site-directed mutagenesis. In general, the technique of site specific mutagenesis is well known in the art as exemplified by publications (Carter, et al., 1985). As will be appreciated, the technique typically employs a phage vector which exists in both a single stranded and double stranded form. Typical vectors useful in site directed mutagenesis include vectors such as the M13 phage (Messing, et al., 1981). These phage are readily commercially available and their use is generally well known to those skilled in the art.
Site directed mutagenesis in accordance herewith is performed by first obtaining a single stranded vector which includes within its sequence the DNA sequence encoding a leader sequence, pelB being used herewith. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically, for example by the method of Cray, et al. (1978). The primer is annealed with the single stranded vector and subjected to DNA polymerizing enzymes such as the E. coli polymerase I Klenow fragment. In order to complete the synthesis of the mutation bearing strand, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. The heteroduplex may be transformed into a bacterial such as E. coli. or S. marcescens cells used herein. Clones are screened using colony hybridization and radiolabelled mutagenic oligonucleotide to identify colonies which contain the mutated plasmid DNA (Carter et al., 1985).
Constructs may also include a xe2x80x9ctagxe2x80x9d useful for isolation and purification of the expressed and secreted polypeptide product. Tags are relatively short DNA segments fused in-frame with a sequence encoding a desired polypeptide, such as the TCR variable domains herein described, which have the function of facilitating detection, isolation and purification. For example, affinity peptides may be encoded by the segments, allowing isolation by selective binding to specific antibodies or affinity resins. Any of a number of tags may be used, including the c-myc tag, (his)6 tag, decapeptide tag (Huse et al., 1989), Flag(trademark) (Immunex) tags and so forth. A number of the tags are also useful for the detection of expressed protein using Western blotting (Ward et al., 1989; Towbin et al., 1978).
(His)6 tags, for example, are preferable for purifying secreted polypeptide products on affinity metal chromatography columns based on metals such as Ni2+. The (his)6 peptide chelates Ni2+ ions with high affinity. Polypeptide products containing these residues at the N or C termini bind to the affinity columns, allowing polypeptide impurities and other contaminants to be washed away as part of the purification process. Polypeptide products can then be eluted from the column with high efficiency using, for example, 250 mM imidazole.
Peptide tags, or linkers, may also be incorporated into the TCR product. For single chain TCR fragments, preferred linker peptides include a 15-mer, for example, (gly4ser)3, or other linkers, such as those described in Filpula and Whitlow (1991).
The invention has been illustrated with prokaryotic host cells, but this is not meant to be a limitation. The prokaryotic specific promoter and leader sequences described herein may be easily replaced with eukaryotic counterparts. It is recognized that transformation of host cells with DNA segments encoding any of a number of T-cell variable domains will provide a convenient means of providing fully functional TCR protein. Both cDNA and genomic sequences are suitable for eukaryotic expression, as the host cell will, of course, process the genomic transcripts to yield functional mRNA for translation into protein.
It is similarly believed that almost any eukaryotic expression system may be utilized for the expression of TCR proteins, e.g., baculovirus-based, glutamine synthase based or dihydrofolate reductase-based systems could be employed. Plasmid vectors would incorporate an origin of replication and an efficient eukaryotic promoter, as exemplified by the eukaryotic vectors of the pCMV series, such as pCMV5.
For expression in this manner, one would position the coding sequences adjacent to and under the control of the promoter. It is understood in the art that to bring a coding sequence under the control of such a promoter, one positions the 5xe2x80x2 end of the transcription initiation site of the transcriptional reading frame of the protein between about 1 and about 50 nucleotides xe2x80x9cdownstreamxe2x80x9d of (i.e., 3xe2x80x2 of) the chosen promoter.
Where eukaryotic expression is contemplated, one will also typically desire to incorporate into the transcriptional unit, an appropriate polyadenylation site (e.g., 5xe2x80x2-AATAAA-3xe2x80x2) if one was not contained within the original cloned segment. Typically, the poly A addition site is placed about 30 to 2000 nucleotides xe2x80x9cdownstreamxe2x80x9d of the termination site of the protein at a position prior to transcription termination.
As used herein the term xe2x80x9cengineeredxe2x80x9d or xe2x80x9crecombinantxe2x80x9d cell is intended to refer to a cell into which a recombinant gene, such as a gene encoding a T-cell receptor variable domain, has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells which do not contain a recombinant gene that is introduced by transfection or transformation techniques. Engineered cells are thus cells having a gene or genes introduced through the hand of man. Recombinantly introduced genes will either be in the form of a cDNA (i.e., they will not contain introns), a copy of a cDNA gene, genomic DNA (with or without introns; for expression in prokaryotic hosts, the DNA should be without introns), or will include DNA sequences positioned adjacent to a promoter not naturally associated with the particular introduced gene.
Generally speaking, it may be more convenient to employ as the recombinant gene a cDNA version of the gene. It is believed that the use of a cDNA gene will provide advantages in that the size of the gene is generally much smaller and more readily employed to transform (or transfect) a targeted cell than a genomic gene, which will typically be up to an order of magnitude larger than the cDNA gene. However, the inventor does not exclude the possibility of employing a genomic version of a particular gene where desired, for expression in mammalian cells. For prokaryotic host cells, constructs without introns will be used, since prokaryotes do not splice introns and exons into functional mRNA.
Suitable host cells useful in the practice of the invention include gram-negative organisms and might include Serratia marcescens, Salmonella typhimurium and similar species. A particularly preferred host cell is E. coli and the several variants of E. coli that are readily available and well known to those of skill in the art.
A particular aspect of the invention is a method for the production of T-cell receptor variable domains. A gram-negative microorganism host cell is transformed with any of the disclosed recombinant vectors cultured in an appropriate bacterial culture medium to produce T-cell receptor variable domains which are subsequently isolated. Culturing typically comprises both a growing and an induction step. Growing is conveniently performed in such media as Luria broth plus 1% glucose, 4xc3x97TY (double strength 2xc3x97TY) plus 1% glucose, minimal media plus casamino acids and 5% w/v glycerol with temperatures in the range of 20xc2x0 C. to about 37xc2x0 C., preferably between 25-30xc2x0 C. All media contains ampicillin at a concentration of 0.1-1 mg/ml; to select bacterial cells which contain the expression plasmid. Induction of expression is typically performed at a point after growth has been initiated, usually after 12-16 hours at 30xc2x0 C. This length of growth results in the cells being in the early stationary phase at the induction stage. If the growth media contains glucose, the cells are pelleted and washed prior to addition of inducer (isopropylthiogalactopyranoside (IPTG) at a concentration of 0.1-1 mM) since glucose inhibits induction or expression. Cells may be grown for shorter periods prior to induction, for example for 6-10 hours, or to the mid-exponential stage of growth. Cells are induced for 5-28 hours. 5-6 hours of induction is a preferred induction time if the protein is to be isolated from the periplasm, since longer induction times result in the protein leaking into the culture supernatant. However, it may be desirable to isolate product from the external medium, in which case one would prefer using longer induction times. Temperatures in the range of 20xc2x0 C. to 37xc2x0 C. may be used as growth and induction temperatures, with 25xc2x0 C. being a preferred induction temperature.
Isolating polypeptide products produced by the microbial host cell and located in the periplasmic space typically involves disrupting the microorganism, generally by such means as osmotic shock, sonication or lysis. Once cells are disrupted, cells or cell debris may be conveniently removed by centrifugation or filtration, for example. The proteins may be further purified, for example, by affinity metallic resin chromatography when appropriate peptide tags are attached to the polypeptide products.
Alternatively, if the induction period is longer than 8 hours (at 25xc2x0 C., for example), so that the protein leaks into the culture supernatant, cells may be removed from the culture by centrifugation and the culture supernatant filtered and concentrated (for example, 10-20 fold). Concentrated supernatant is then dialyzed against phosphate buffered saline and separation achieved by column chromatography, such as affinity or adsorption chromatography. An example is separation through Ni2+-NTA-agarose to separate appropriately tagged proteins such as those carrying a (his)6 tag. When these tags are used in the construction of an expression vector, histidine tags are particularly preferred as they facilitate isolation and purification on metallic resins such as Ni+2-NTA agarose.
Also contemplated within the scope of the invention are the recombinant T-cell receptor single-chain variable domain products. These include single chain heterodimers comprising the variable domains Vxcex1, Vxcex2, Vxcex3 and V67 . However, it will be appreciated that modification and changes may be made in the composition of these domains, for example by altering the underlying DNA, and still obtain a molecule having like or otherwise desirable characteristics.
In general, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or receptor sites. Since it is the interactive capacity and nature of a protein that defines that protein""s biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a protein with like or even countervailing properties (e.g., antagonistic v. agonistic). It is thus contemplated by the inventor that various changes may be made in the coding sequence for the T-cell variable domains without appreciable loss of the biological utility or activity of the encoded protein. It may even be possible to change particular T-cell receptor variable domain residues and increase the interactive ability, i.e., binding affinity of the variable domains for cognate peptide MHC complex.
In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte et al., 1982). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (xe2x88x920.4); threonine (xe2x88x920.7); serine (xe2x88x920.8); tryptophan (xe2x88x920.9); tyrosine (xe2x88x921.3); proline (xe2x88x921.6); histidine (xe2x88x923.2); glutamate (xe2x88x923.5); glutamine (xe2x88x923.5); aspartate (xe2x88x923.5); asparagine (xe2x88x923.5); lysine (xe2x88x923.9); and arginine (xe2x88x924.5).
It is believed that the relative hydropathic character of the amino acid may play a role in determining the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid may be substituted by another amino acid having a similar hydropathic index and still obtain a biological functionally equivalent protein. In such changes, the substitution of amino acids whose hydropathic indices are within xc2x12 is preferred, those which are within xc2x11 are particularly preferred, and those within xc2x10.5 are even more particularly preferred. It is also conceivable that it may be possible to increase the binding affinity of a T-cell receptor variable domain by changing an amino acid to another which is quite different in hydrophobicity. This may not have an adverse effect on the structure of the protein, since the residues which interact with peptide-MHC complexes are believed to be located in the exposed hypervariable loops of the V domains.
Substitution of like amino acids can also be made on the basis of hydrophilicity, particularly where the biological functional equivalent protein or peptide thereby created is intended for use in immunological embodiments. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.
As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0xc2x11); glutamate (+3.0xc2x11); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); (0xc2x11); threonine (xe2x88x920.4); alanine (xe2x88x920.5); histidine (xe2x88x920.5); cysteine (xe2x88x921.0); methionine (xe2x88x921.3); valine (xe2x88x921.5); leucine (xe2x88x921.8); isoleucine (xe2x88x921.8); tyrosine (xe2x88x922.3); phenylalanine (xe2x88x922.5); tryptophan (xe2x88x923.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still, although not always, obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within xc2x12 is preferred, those which are within xc2x11 are particularly preferred, and those within xc2x10.5 are even more particularly preferred.
Amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
While discussion has focused on functionally equivalent polypeptides arising from amino acid changes, it will be appreciated that these changes may be effected by alteration of the encoding DNA; taking into consideration also that the genetic code is degenerate and that two or more codons may code for the same amino acid.
As illustrated herein, transformed E. coli host cells will provide good yields of T-cell receptor variable domains. The yields of about 1-2 mg/L for Vxcex1, 0.1-0.2 mg/L for Vxcex2 and 0.5-1 mg/L for the single chain Vxcex1Vxcex2 heterodimer may be readily scaled up to produce relatively large quantities of these TCR domains in a matter of days, employing, for example, a (his)6 tag for affinity purification with the Ni2+-NTA-agarose. Thus the expression system will provide a valuable source of soluble TCR protein for use in immunizations for the generation of anti-clonotypic antibodies, useful, for example, in passive immunization for the treatment of disease. As an example, TCRs expressed on the surface of leukemic T-cells could be expressed as soluble domains and used in immunization to generate anti-TCR antibodies. Such antibodies could be used as targeting reagents in the therapy of T-cell leukemias. It is also conceivable that such soluble TCRs (derived from pathogenic T cells) may be used in vaccination to generate a specific anti-TCR response in vivo for the therapy of autoimmune diseases in a similar way to that reported using peptides derived from TCR V-regions (Vandenbark, et al., 1989; Howell, et al., 1989; Offner, et al., 1991). Moreover, recombinant Vxcex1, Vxcex2, V67, Vxcex3, single chain Vxcex1Vxcex2 fragments, domains, or even subfragments thereof, are potentially useful for mapping the TCR residues which are functionally important in binding peptide-MHC complexes.
The present invention shows that scTCR (from Vxcex1 and Vxcex2) derived from the 1934.4 T-cell hybridoma (Wraith et al., 1989) is secreted into the periplasm and may be purified in yields of approximately 0.5-1 mg/L culture using Ni2+-NTA-agarose. FIG. 2 shows an SDS polyacrylamide gel analysis of the purification of this protein. For the scTCR in particular, lower growth and induction temperatures of 25-30xc2x0 C. resulted in higher expression yields. Even higher expression may be achieved with modifications to growth medium and temperature, as recognized by those of skill in the art. For example, lower growth and induction temperatures were found to enhance expression of other recombinant proteins in E. coli (Takagi et al., 1988).
Purification of the herein described murine TCR variable domains may be achieved in many ways, including chromatography, density gradient centrifugation and electrophoretic methods. A particular example of scTCR purification employs an affinity column, made by linking the monoclonal antibody KJ16 (specific for murine Vxcex28: Kappler et al., 1988) to sepharose. For the 1934.4 derived scTCR, purification with this affinity column indicated that the epitope recognized by this monoclonal antibody is in the correct conformational state in the recombinant scTCR.
A rapid method for the production of soluble, heterodimeric TCRs, as presented in the present disclosure, may be readily extended to the production of soluble TCRs of different specificities, derived from other species such as man. This opens up new avenues for immunotherapy and diagnosis, particularly in relation to T cell mediated autoimmune diseases and T-cell leukemias. Also contemplated is the use of random in vitro mutagenesis to alter the residues of recombinant TCR fragments, and to express these fragments as either soluble proteins as disclosed herein or on the surface of bacteriophage (McCafferty et al., 1990). Mutants binding with higher affinity to peptide-MHC complexes may be screened for or selected for using solid surfaces coated with antigen presenting cells and cognate peptide. Such higher affinity mutants would have a large number of applications, for example, in therapy of autoimmune disease as blocking reagents. The TCR fragments are produced in sufficient quantities for a wide variety of tests and studies including, for example, high resolution structural analyses with NMR and X-ray diffraction.